It has long been recognized that the ability of tumor tissues to metastasize constitutes a major portion of the life-threatening aspects of malignancy. Metastasis is the growth of secondary tumors at sites different from the primary tumor. Thus, despite surgical removal of the primary tumor, it may not be possible to arrest the progress of this condition. An understanding of the mechanism whereby metastasis occurs will be crucial to the development of protocols whereby the growth of secondary tumors can be controlled. In order to understand the mechanism of metastasis, it will be necessary to provide a model which permits identification of small numbers of tumor cells against a background of many host cells so that secondary tumor emboli and micrometastases can be observed over the course of real time.
Others have demonstrated extravasation and initial seeding steps in tumor metastasis in vitro using externally fluorescently labeled tumor cells. Khokha, R. et al., Cancer Metastasis Rev (1995) 14:279-301; Koop, S. et al., Cancer Res (1995) 55:2520-2523. Further, Margolis, L. B. et al., In Vitro Cell Dev Biol (1995) 31:221-226 was able to visualize the migration of externally fluorescently labeled lung tumor cells in host mouse lung in histoculture. In all cases, however, long-term observation was not possible due to the limitation of exogenous fluorescent labels. Retroviral transfer of a green fluorescent protein (GFP) gene has been shown to result in stable transfectants of human cancer cells in vitro (Levy, J. P. et al., Nature Biotechnol (1996) 14:610-614), as well as of hematopoietic cells (Grignani, F. et al. Cancer Res (1998) 58:14-19 and by Cheng, L. et al. Gene Therapy (1997) 4:1013-1022).
Attempts have been made to provide such a model using the .beta.-galactosidase gene as a marker (Lin, W. C. et al., Cancer Res (1990) 50:2808-2817; Lin, W. C. et al., Invasion and Metastasis (1992) 12:197-209). However, this marker has not proved satisfactory, as fresh or processed tissue cannot be used. The present invention provides a marker which permits visualization of tumor invasion and micrometastasis formation in viable fresh tissue. In addition, by providing suitable contrast media, the method of the invention can be adapted to visualize angiogenesis in established and growing tumors. The methods of the invention can be applied not only to models of tumor growth and metastasis, but, through the use of retroviral vectors, can be employed to obtain clinical data in human subjects bearing tumors.
The present invention utilizes green fluorescent protein (GFP) as a marker. Heterologous expression of this protein, principally to monitor expression of fused DNA, was disclosed in U.S. Pat. No. 5,491,084. This document describes the expression of GFP in E. coli and C. elegans and postulates that cells in general can be modified to express GFP. Such expression, according to this document, permits not only a method to monitor expression of fused DNA, but also a means of monitoring protein localization within the cell.
The aspect of the invention which provides a metastatic model has been reported and described in a series of publications. Chishima, T. et al. Cancer Research (1997) 57:2042-2047 describe the construction of a dicistronic vector containing the gene for humanized green fluorescent protein (GFP) and dihydrofolate reductase (DHFR). This vector was transfected into CHO-K1 cells to obtain clone-38. Clone-38 showed stable GFP expression which was maintained in the presence of methotrexate (MTX). Clone-38 cells were injected into mice to obtain tumor fragments which were then implanted by surgical orthotopic implantation (SOI) on the ovarian serosa in nude mice. Metastasis could be followed in this model.
Chishima, T. et al. Proc Natl Acad Sci USA (1997) 94:11573-11576 describe the preparation of clone-26 by transfection of Anip 973 human lung adenocarcinoma cells with the codon optimized hGFP-S65T clone obtained from Clontech. Clone-26 was injected intravenously into nude mice and the resulting tumors were followed in histoculture.
Chishima, T. et al. Clin Exp Metastasis (1997) 15:547-552 and Chishima, T. et al. Anticancer Res (1997) 17:2377-2384 describe similar work with clone-26 wherein the cells were inoculated subcutaneously into nude mice resulting in a visualizable tumor which was then implanted into the visceral pleura of nude mice by SOI. Metastases were observed in this model as well.
Chishima, T. et al. In Vitro Cell Dev Biol (1997) 33:745-747 describe histoculture of clone-26 and visualization of growth using the fluorescence emitted by GFP.
Yang, M., et al., Cancer Res (1998) 58:4217-4221 describe transduction of the human lung cancer cell line H460 with a retroviral expression vector containing enhanced GFP to obtain a stable high-GFP-expressing clone. Cells from this cell line were injected into nude mice and the resulting subcutaneously growing labeled tumors were transplanted by SOI into the left lung of nude mice. Fluorescence could then be observed from the metastases in the collateral lung, pleural membrane and throughout the skeletal system.
Yang, M., et al., Cancer Res (In Press) report similar studies using a model for prostate tumor and showing fluorescence throughout the skeletal system in nude mice.
The contents of the foregoing publications are incorporated herein by reference.
In addition to the foregoing, Cheng, L., et al., Gene Therapy (1997) 4:1013-1022, describe the modification of hematopoietic stem cells using the GFP gene under control of a retroviral promoter. Although the authors state that human stem cells are transfected with this system only with difficulty, by using an enhanced form of the GFP, satisfactory brightness could be achieved.
In addition, Grignani, F., et al., Cancer Res (1998) 58:14-19, report the use of a hybrid EBV/retroviral vector expressing GFP to effect high-efficiency gene transfer into human hematopoietic progenitor cells.
Vectors containing various modified forms of GFP to provide various colors are marketed by Clontech. The Clontech vectors intended for mammalian cell expression place the GFP under control of the cytomegalovirus (CMV) promoter.